Electrical, Optical and Sensoric Properties of Organic Semiconductors

Existuje velký zájem o levné, citlivé a selektivní senzory. V této práci popisujeme využití rozpustných substituovaných ftalocyaninů jako citlivých materiálů pro detekci řady plynů. Studovali jsme optické, elecktrické a senzorové vlastnosti několika ftalocyaninů. Byl popsán mechanizmus interakce tenkých vrstev ftalocyaninů s různými studovanými plyny. Zjistilo se, že ftalocyaniny substituované sulfo-skupinami jsou velmi citlivé na vodní páru. Sulfonamidicky substituované ftalocyaniny jsou vhodné pro detekci oxidu dusičitého a těkavých organických rozpouštědel. Při vyšších teplotách tert-butylový ftalocyanin je vhodný pro detekci nízkých NO2 koncentrací. Experimentální výsledky byly využity pro konstrukci nových typů komerčních senzorů pro detekci NO2, etanolu a vlhkosti.There is big interest in cheap, sensitive and selective gas sensors. In this work, substituted soluble phthalocyanines are proposed as a sensing materials for several gases. Optical, electrical and gas sensing properties of several phthalocyanines were studied and the mechanisms of their interaction with several analyte gases are described. It was found, that sulfo-substituted Pcs has good sensitivity to humidity. Sulfonamide-substituted phthalocyanines are promising for nitrogen dioxide and volatile organic compounds detection. tert-Butyl-substituted phthalocyanines are sensitive to NO2 under higher temperature and seems to be used for environmental monitoring. Commercial gas sensors for NO2, ethanol and humidity were successfully created.

Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

Background: Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. Principal Findings: We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L−1 of culture from E. coli with specific activities of 1000 U (U = 1 µmol hydrogen evolved mg−1 min−1). Significance: The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.United States. Dept. of Energy. (contract DE-AC36-08-GO28308

Electrical, Optical and Sensoric Properties of Organic Semiconductors

There is big interest in cheap, sensitive and selective gas sensors. In this work, substituted soluble phthalocyanines are proposed as a sensing materials for several gases. Optical, electrical and gas sensing properties of several phthalocyanines were studied and the mechanisms of their interaction with several analyte gases are described. It was found, that sulfo-substituted Pcs has good sensitivity to humidity. Sulfonamide-substituted phthalocyanines are promising for nitrogen dioxide and volatile organic compounds detection. tert-Butyl-substituted phthalocyanines are sensitive to NO2 under higher temperature and seems to be used for environmental monitoring. Commercial gas sensors for NO2, ethanol and humidity were successfully created

SDS-PAGE analysis of Fd-HydA1 expression, TEV digestion and HydA1 purification.

<p>(A). A comparison of the expression levels of N-terminal and C-terminal Fd-HydA1 fusions by separation on SDS-PAGE. Lanes show (left to right) protein size marker, SM, with sizes in kDa, and increasing amounts (0.5-to-4 µl) of total protein from cells expressing either the C-terminal (Fd C) or N-terminal (Fd N) Fd-HydA1 fusions. The location of ∼60 kDa Fd-HydA1 fusion, and 53 kDa HydG as an internal loading control are marked. The difference of Fd position on Fd-HydA1 expression levels was evident as a more intense brown color of the cell-lysates (inset image, top) harboring the N-terminal (right) versus C-terminal (left) Fd fusion. A Western Blot performed with antibodies to the StrepII-tag is shown (inset image, bottom) where the N-terminal fusion (right) exhibits a more intense band than the C-terminal fusion (left). (B). Analysis of TEV digestions of the N-terminal Fd-HydA1 fusion (30 µg) mixed with 1, 2, 4, or 8 µg of TEV (lanes 1–4, respectively). Lanes 2 and 3 show a partial digestion. The locations of Fd-HydA1 (60 kDa), HydA1 (50 kDa) and Fd (10 kDa) are indicated on the right. The optimal amount of TEV for complete digestion was 4 µg (shown in lane 3). Protein size-marker, SM, with sizes in kDa. (C). TEV digestions of DEAE pooled fractions containing Fd-HydA1 and separated on SDS-PAGE. The same w/w ratio of TEV to Fd-HydA1 was used as for purified Fd-HydA1 (4 µg TEV with 30 µg of DEAE pool). Lane 1, complete TEV digest; Lane 2, partial TEV digest; Lane 3, no TEV. Bands corresponding to Fd-HydA1 (60 kDa), HydA1 (50 kDa) and Fd (10 kDa) are identified on the right. HydG is identified as a loading control. (D). Analysis of HydA1 purity by SDS-PAGE. Protein size marker, SM, with sizes in kDa. Lanes 1–4 contain 1.4, 4, 6.8, and 12.3 µg, respectively, of purified HydA1 at >99% purity.</p

HydA1 purification yields and specific activities from various expression hosts.

a<p>HydA1 specific activities for hydrogen evolution by MV assay; 1 U = µmol hydrogen min⁻¹ mg⁻¹.</p>b<p>HydA1 mg L⁻¹ of cell culture.</p>c<p>Iron content as mol iron (mol HydA1)⁻¹; NR, not reported.</p

A Fluorescent Readout for the Oxidation State of Electron Transporting Proteins in Cell Free Settings

Pathways involving sequential electron transfer between multiple proteins are ubiquitous in nature. Here, we demonstrate a new class of fluorescent protein-based reporters for monitoring electron transport through such multistage cascades, specifically those involving ferredoxin-like electron transporters. We created protein fusions between mammalian Adrenodoxin (Adx) and plant Ferredoxin (Fdx) with fluorescent proteins of different colors and found that the fluorescence of such fusions is highly sensitive to the redox state of the electron transporter. The increase in fluorescence from the oxidized to the reduced state was inversely proportional to the linker length between the fusion partners. We first used our approach to quantitatively characterize electron transfer from NADPH through Adrenodoxin Reductase (AdR) to Adrenodoxin (Adx). Our data allowed us to build a detailed mathematical model of this mitochondrial electron transfer chain and validate previously proposed mechanisms. Then, we showed that an Adx-GFP fusion could serve as a sensor for the activity of bacterial Type I Cytochrome P450s (CYPs), a very large class of enzymes with important roles in biotechnology. We further showed that fluorescence of a direct fusion between CYP and GFP was sensitive to CYP activity, suggesting that our approach is applicable to an even broader class of proteins, which undergo a redox state change during their work cycle

Influence of growth factors on the <i>in vitro</i> hydrogen evolution activities in cells harboring pHydEFd-HydA1 and pHydFHydG.

<p>Hydrogenase activities (reported in units of nmol hydrogen evolved, ml⁻¹ of culture, min⁻¹) in solubilized whole-cells were measured as described in materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035886#s4" target="_blank">methods</a>. (A). The effect of the shaker rotation speed (RPM) during the aerobic growth phase was tested for 100 ml or 1 L culture volumes grown in. 250 ml or 2 L baffled flasks, respectively. The optimal rate was for both culture volumes was ∼300–350 RPM. (B). The effect of ferric ammonium citrate (FEC) concentration. Optimal levels were 2–2.5 mM. (C). Effect of IPTG concentration. Optimal levels of IPTG were 0.4 mM, lower than the 1.5 mM used previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035886#pone.0035886-Evans1" target="_blank">[13]</a>. (D). Effect of substituting Ampicillin with Carbenicillin during all stages of growth. Addition of Carbenicillin at 200 µg ml⁻¹ was required for peak hydrogenase activity levels.</p

Buffers used for protein purification.

<p>Buffers used for protein purification.</p